This Project: This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.
Background Information: ACE which is short for Angiotensin I-converting enzyme, is a zync metallopeptidase that converts angiotensin I into angiotensin II. Angiotensin II a vasoactive and aldosterone-stimulating peptide [6]. The protein also deactivates bradykinin, commonly known as BK, by removing two C-terminal dipeptides [5]. The major features of ACE are the results of a gene duplication [4]. It is made up of two homologous domains, N- and C- [5]. ACE is thought to have other physiological roles because of its wide enzymatic specificity and wide distribution in animals[6]. Genes similar to ACE are widely distributed and common in nature [4]. ACE can be found in biological fluids such as plasma and amniotic or seminal fluids [6]. Two forms of ACE exist in humans [5]. The first is a somatic ACE which is around 170 kDA, and it is found in endothelial, epithelial, and neuronal cells [5]. The second is a smaller testicular isoform around 100 kDA in size and it is found in germinal cells [5]. The Somatic form of ACE is involved in regulating the cardiovascular system, and the Germinal form of ACE is involved in reproduction for males [5]. Both the somatic and germinal ACE forms are generated by the initiation of transcription from different start sites under the control of two separate promoters [5].
Figure 1. Shows the protein ACE and how it regulates blood pressure, fluid homeostasis, electrolyte balance, and male fertility.
Methods/Approach: The amino acid sequence of the gene was found using Ensembl databases by using the gene’s Ensemble ID (ENSP00000290866). This was done by entering the Ensemble ID, and then selecting ‘human’ as the species to search against. A transcript associated with the specific protein ID was chosen. BLASTp was then performed on the amino acid sequence. Once a good match was found from the BLAST results, similar sequences in other species were found using the NCBI Blast program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Homologues were found in particular species by selecting the ‘protein blast’ option and running the search. The FASTA format of the sequence was inserted into the ‘query sequence’ box, ‘blastp’ was the selected tab, and the sequence was “BLASTed.” The whale shark database was imported from the Galaxy server (whaleshark.georgiaaquarium.org). Imported data appeared as a green numbered item. The human sequence of the gene was entered as the query sequence by selecting ‘get data’ and then ‘upload data.’ The FASTA format of the sequence was then entered into the text box and then executed. Once the whale shark database and the query sequence were both uploaded to the history, a BLAST search was performed by clicking ‘NCBI Blast+.’ The pasted sequence was chosen as the query and the ‘FASTA file from history’ was chosen as the subject database. E-value and length of alignment were used to determine if any of the results were significant hits in the whale shark database. Once a significant BLAST hit was discovered, its sequence was retrieved. ‘Whale-shark.predicted-genes’ database was selected and the sequence ID of the predicted gene was entered. Phylogenetic Tree To align sequences, Clustal algorithm was used. The sequence alignment was then used to create the phylogenetic tree.
ACE in the Whale Shark The human ACE protein sequence was used as query against the whale shark protein database and some of the results are shown in Table 1. There were numerous hits, and the most significant had an E-value of 1e-88. The five most significant hits are listed in Table 1. The top five hits, according to e-value and percent identity, were BLASTED against the human protein database using NCBI BLASTp.
Table 1. Top Five Hits of the Human ACE Protein against the Whale Shark Protein Database
Whale Shark ID
e-value
Alignment Length
Predicted Protein Length
% Identity
g36554
1.00E-88
162
1306
87.65
g40202
4.00E-56
114
1306
83.33
g30771
2.00E-19
85
1306
56.42
g41045
8.00E-04
44
1306
54.55
g16556
7.00E-04
39
1306
51.28
Protein Domains: The top five hits of the whale shark predicted protein ACE contained putative M2_ACE. Within each of the five hits, there were two homologous sequences that each contained potential Zn-binding regions and catalytic sites as shown in Figure 2. The homologous regions are about half the size of the whole ACE protein, and they suggest that the current and modern version of the ACE gene is the duplicated product of the ancestor gene [3]. The potential matches that were found while blasting the ACE protein shows that each match contains M2_ACE as well as Peptidase_M2. M2_ACE is a dipeptidase that converts or catalyzes the conversion of angiotensin I to angiotensin II. This gene also encodes two versions of enzymes in humans: ubiquitous somatic ACE and sperm-specific germinal ACE. The somatic ACE has two sites of reactivity while the germinal, which is widely unknown , only has one. The Peptidase_M2 gene promotes an exact same function as the M2_ACE however the most notable difference is that the members of this domain contain a tandem duplication and catalytically active amino acid groups
Figure 2. Putative domains of whale shark ACE best hit predicted proteins. The five best hit whale shark predicted proteins contain putative M2_ACE as predicted by NCBI BLAST server analyses. Peptidase Gluzincin Family includes many different peptidases. “All peptidases in this family bind a single catalytic zinc ion which is tetrahedrally co- ordinated by three amino acid ligands and a water molecule that forms the nucleophile on activation during catalysis” (NCBI).
Orthologs The human ACE sequence (ENSP00000290866) was used as query in NCBI BLAST searches against individual species' protein databases. These species consisted of Elephant Shark, Clawed Frog, and Zebra Fish. The best hits against the human ACE sequence are shown in Table 2. The species, description, max score, total score, query cover, e-value, identity, and accession are listed in the table.
Table 2. Whale Shark ACE Sequence BLAST against Other Species
Phylogeny The top five hits from the protein database search for the ACE protein as query were used to create a phylogenetic tree. It is clear from this tree that the whale shark predicted proteins are dispersed throughout the tree at various points. This means that they do not necessarily have a high degree of similarity (Figure 3).
Figure 3. Phylogenetic tree of the best hits of ACE and other species that it is compared to.
Conclusion: Based on the phylogenetic tree and the results of our blasts, the evidence leads us to believe that the Whale Shark does indeed have some form of the ACE gene present within its genome. When looking at the cross referenced genes from whale shark to human and whale shark to other species, the genes indeed had the same domain families and therefore we believe that ACE should serve a similar function in the whale shark in maintaining homeostasis, regulating blood pressure, and electrolyte balance. Genes in the whale shark such as g30771 and g36554 assert a clear similarity between the ACE gene in humans and whale sharks due to how close it is on the phylogenetic tree and the fact that these genes have high percent identities and query coverages. For some future research, one might consider looking at other aspects of clear differences in these genes from the human genome, and how these differences change the exact function of each gene. This experiment could have been improved if we possibly used more species for comparison and possibly if there was more research that had been done on the function of genes in whale sharks, but unfortunately this is a brand new finding of research and there is much more to be discovered.
References:
1)"ACE Gene." - GeneCards. Weizmann Institute of Science, n.d. Web. 14 Apr. 2015.
2)"ACE Gene." Genetics Home Reference. N.p., May 2013. Web. 14 Apr. 2015.
3) Bernstein, K. E., B. M. Martin, A. S. Edwards, and E. A. Bernstein. "Mouse Angiotensin-converting Enzyme Is a Protein Composed of Two Homologous Domains." The Journal of Biological Chemistry (1989): n. pag. Www.jbc.org. Web. 10 Apr. 2015. <http://www.jbc.org/content/264/20/11945.short>.
4) Bernstein, K. E., F. S. Ong, W.-L. B. Blackwell, K. H. Shah, J. F. Giani, R. A. Gonzalez-Villalobos, X. Z. Shen, and S. Fuchs. "A Modern Understanding of the Traditional and Nontraditional Biological Functions of Angiotensin-Converting Enzyme." Pharmacological Reviews 65.1 (2012): 1-46. Web. 10 Apr. 2015.
5) Michaud, A., K. Acharya, G. Masuyer, N. Quenech'du, O. Gribouval, V. Moriniere, M. Gubler, and P. Corvol. "Absence of Cell Surface Expression of Human ACE Leads to Perinatal Death." Absence of Cell Surface Expression of Human ACE Leads to Perinatal Death. Oxford Journals, 24 Oct. 2013. Web. 12 Apr. 2015.
6) Rigat, B., C. Hubert, F. Alhenc-Gelas, F. Cambien, P. Corvol, and F. Soubrier. "An Insertion/Deletion Polymorphism in the Angiotensin I-converting Enzyme Gene Accounting for Half the Variance of Serum Enzyme Levels." The American Society for Clinical Investigation, Inc 86 (1990): 1343-346. Print. 5)Tiret, L et al. “Evidence, from Combined Segregation and Linkage Analysis, That a Variant of the Angiotensin I-Converting Enzyme (ACE) Gene Controls Plasma ACE Levels.” American Journal of Human Genetics 51.1 (1992): 197–205. Print.
This Project:
This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.
Background Information:
ACE which is short for Angiotensin I-converting enzyme, is a zync metallopeptidase that converts angiotensin I into angiotensin II. Angiotensin II a vasoactive and aldosterone-stimulating peptide [6]. The protein also deactivates bradykinin, commonly known as BK, by removing two C-terminal dipeptides [5].
The major features of ACE are the results of a gene duplication [4]. It is made up of two homologous domains, N- and C- [5]. ACE is thought to have other physiological roles because of its wide enzymatic specificity and wide distribution in animals[6]. Genes similar to ACE are widely distributed and common in nature [4]. ACE can be found in biological fluids such as plasma and amniotic or seminal fluids [6]. Two forms of ACE exist in humans [5]. The first is a somatic ACE which is around 170 kDA, and it is found in endothelial, epithelial, and neuronal cells [5]. The second is a smaller testicular isoform around 100 kDA in size and it is found in germinal cells [5]. The Somatic form of ACE is involved in regulating the cardiovascular system, and the Germinal form of ACE is involved in reproduction for males [5]. Both the somatic and germinal ACE forms are generated by the initiation of transcription from different start sites under the control of two separate promoters [5].
Figure 1. Shows the protein ACE and how it regulates blood pressure, fluid homeostasis, electrolyte balance, and male fertility.
Methods/Approach:
The amino acid sequence of the gene was found using Ensembl databases by using the gene’s Ensemble ID (ENSP00000290866). This was done by entering the Ensemble ID, and then selecting ‘human’ as the species to search against. A transcript associated with the specific protein ID was chosen. BLASTp was then performed on the amino acid sequence. Once a good match was found from the BLAST results, similar sequences in other species were found using the NCBI Blast program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Homologues were found in particular species by selecting the ‘protein blast’ option and running the search. The FASTA format of the sequence was inserted into the ‘query sequence’ box, ‘blastp’ was the selected tab, and the sequence was “BLASTed.”
The whale shark database was imported from the Galaxy server (whaleshark.georgiaaquarium.org). Imported data appeared as a green numbered item. The human sequence of the gene was entered as the query sequence by selecting ‘get data’ and then ‘upload data.’ The FASTA format of the sequence was then entered into the text box and then executed. Once the whale shark database and the query sequence were both uploaded to the history, a BLAST search was performed by clicking ‘NCBI Blast+.’ The pasted sequence was chosen as the query and the ‘FASTA file from history’ was chosen as the subject database. E-value and length of alignment were used to determine if any of the results were significant hits in the whale shark database. Once a significant BLAST hit was discovered, its sequence was retrieved. ‘Whale-shark.predicted-genes’ database was selected and the sequence ID of the predicted gene was entered.
Phylogenetic Tree
To align sequences, Clustal algorithm was used. The sequence alignment was then used to create the phylogenetic tree.
ACE in the Whale Shark
The human ACE protein sequence was used as query against the whale shark protein database and some of the results are shown in Table 1. There were numerous hits, and the most significant had an E-value of 1e-88. The five most significant hits are listed in Table 1. The top five hits, according to e-value and percent identity, were BLASTED against the human protein database using NCBI BLASTp.
Table 1. Top Five Hits of the Human ACE Protein against the Whale Shark Protein Database
Protein Domains:
The top five hits of the whale shark predicted protein ACE contained putative M2_ACE. Within each of the five hits, there were two homologous sequences that each contained potential Zn-binding regions and catalytic sites as shown in Figure 2. The homologous regions are about half the size of the whole ACE protein, and they suggest that the current and modern version of the ACE gene is the duplicated product of the ancestor gene [3]. The potential matches that were found while blasting the ACE protein shows that each match contains M2_ACE as well as Peptidase_M2. M2_ACE is a dipeptidase that converts or catalyzes the conversion of angiotensin I to angiotensin II. This gene also encodes two versions of enzymes in humans: ubiquitous somatic ACE and sperm-specific germinal ACE. The somatic ACE has two sites of reactivity while the germinal, which is widely unknown , only has one. The Peptidase_M2 gene promotes an exact same function as the M2_ACE however the most notable difference is that the members of this domain contain a tandem duplication and catalytically active amino acid groups
Figure 2. Putative domains of whale shark ACE best hit predicted proteins. The five best hit whale shark predicted proteins contain putative M2_ACE as predicted by NCBI BLAST server analyses. Peptidase Gluzincin Family includes many different peptidases. “All peptidases in this family bind a single catalytic zinc ion which is tetrahedrally co- ordinated by three amino acid ligands and a water molecule that forms the nucleophile on activation during catalysis” (NCBI).
Orthologs
The human ACE sequence (ENSP00000290866) was used as query in NCBI BLAST searches against individual species' protein databases. These species consisted of Elephant Shark, Clawed Frog, and Zebra Fish. The best hits against the human ACE sequence are shown in Table 2. The species, description, max score, total score, query cover, e-value, identity, and accession are listed in the table.
Table 2. Whale Shark ACE Sequence BLAST against Other Species
Phylogeny
The top five hits from the protein database search for the ACE protein as query were used to create a phylogenetic tree. It is clear from this tree that the whale shark predicted proteins are dispersed throughout the tree at various points. This means that they do not necessarily have a high degree of similarity (Figure 3).
Figure 3. Phylogenetic tree of the best hits of ACE and other species that it is compared to.
Conclusion: Based on the phylogenetic tree and the results of our blasts, the evidence leads us to believe that the Whale Shark does indeed have some form of the ACE gene present within its genome. When looking at the cross referenced genes from whale shark to human and whale shark to other species, the genes indeed had the same domain families and therefore we believe that ACE should serve a similar function in the whale shark in maintaining homeostasis, regulating blood pressure, and electrolyte balance. Genes in the whale shark such as g30771 and g36554 assert a clear similarity between the ACE gene in humans and whale sharks due to how close it is on the phylogenetic tree and the fact that these genes have high percent identities and query coverages. For some future research, one might consider looking at other aspects of clear differences in these genes from the human genome, and how these differences change the exact function of each gene. This experiment could have been improved if we possibly used more species for comparison and possibly if there was more research that had been done on the function of genes in whale sharks, but unfortunately this is a brand new finding of research and there is much more to be discovered.
References:
1)"ACE Gene." - GeneCards. Weizmann Institute of Science, n.d. Web. 14 Apr. 2015.
2)"ACE Gene." Genetics Home Reference. N.p., May 2013. Web. 14 Apr. 2015.
3) Bernstein, K. E., B. M. Martin, A. S. Edwards, and E. A. Bernstein. "Mouse Angiotensin-converting Enzyme Is a Protein Composed of Two Homologous Domains." The Journal of Biological Chemistry (1989): n. pag. Www.jbc.org. Web. 10 Apr. 2015. <http://www.jbc.org/content/264/20/11945.short>.
4) Bernstein, K. E., F. S. Ong, W.-L. B. Blackwell, K. H. Shah, J. F. Giani, R. A. Gonzalez-Villalobos, X. Z. Shen, and S. Fuchs. "A Modern Understanding of the Traditional and Nontraditional Biological Functions of Angiotensin-Converting Enzyme." Pharmacological Reviews 65.1 (2012): 1-46. Web. 10 Apr. 2015.
3)"Basic Local Alignment Search Tool." BLAST:. N.p., n.d. Web. 14 Apr. 2015.
4)"Ensembl Genome Browser." Ensembl Genome Browser. N.p., n.d. Web. 14 Apr. 2015.
5) Michaud, A., K. Acharya, G. Masuyer, N. Quenech'du, O. Gribouval, V. Moriniere, M. Gubler, and P. Corvol. "Absence of Cell Surface Expression of Human ACE Leads to Perinatal Death." Absence of Cell Surface Expression of Human ACE Leads to Perinatal Death. Oxford Journals, 24 Oct. 2013. Web. 12 Apr. 2015.
6) Rigat, B., C. Hubert, F. Alhenc-Gelas, F. Cambien, P. Corvol, and F. Soubrier. "An Insertion/Deletion Polymorphism in the Angiotensin I-converting Enzyme Gene Accounting for Half the Variance of Serum Enzyme Levels." The American Society for Clinical Investigation, Inc 86 (1990): 1343-346. Print.
5)Tiret, L et al. “Evidence, from Combined Segregation and Linkage Analysis, That a Variant of the Angiotensin I-Converting Enzyme (ACE) Gene Controls Plasma ACE Levels.” American Journal of Human Genetics 51.1 (1992): 197–205. Print.
7) Galaxy/ Whale Shark. Coca Cola/ Georgia Aquarium, n.d. Web. 14 Apr. 2015. <http://whaleshark.georgiaaquarium.org/>.
8) Multiple Sequence Alignment by CLUSTALW. CLUSTALW, n.d. Web. 14 Apr. 2015. <http://www.genome.jp/tools/clustalw/>.
9) Image Link: http://journal.chemistrycentral.com/content/3/1/11/figure/F3?highres=y